This invention relates to the determination of live and dead encysted protozoa in a population of protozoa. In particular, the invention relates to the use of vital dyes to determine the viability of sporocysts in a sample and then correlating the extent of staining in the sample with the viability of protozoa in the population from which the sample was obtained.
Coccidiosis is a disease of various animals in which the intestinal mucosa is invaded and damaged by a protozoa of the subclass Coccidia. The economic effects of coccidiosis can be especially severe in the poultry industry where intensive housing of birds favors the spread of the disease. Infection by coccidial protozoa is, for the most part, species specific. Numerous species, however, can infect a single host. For example, there are seven species of Coccidia which infect chickens, six of which are considered to be moderately to severely pathogenic.
The life cycle of the coccidial parasite is complex. For example, protozoa of the genera Eimeria, Isospora, Cystoisospora, or Cryptosporidium typically only require a single host to complete their life cycle, although Cystoisospora may utilize an intermediate host. Under natural conditions, the life cycle begins with the ingestion of sporulated oocysts from the environment. When sporulated oocysts are ingested by a susceptible animal, the wall of the sporulated oocyst is broken in order to release the sporocysts inside. In poultry, the release of the sporocyst is the result of mechanical disruption of the sporulated oocyst in the gizzard. Within the sporocysts are the sporozoites which are the infective stage of the organism. In poultry, the breakdown of the sporocyst coat and release of the sporozoites is accomplished biochemically through the action of chymotrypsin and bile salts in the small intestine. Once released, the sporozoites invade the intestinal mucosa or epithelial cells in other locations. The site of infection is characteristic of the species involved. For example, in the genus Eimeria, E. tenella is localized in the ceca; E. necatrix is found in the anterior and middle portions of the small intestine; E. acervulina and E. praecox occur in the upper half of the small intestine; E. brunetti occurs in the lower small intestine, rectum, ceca, and cloaca; while E. mitis is found in the lower small intestine.
Once inside the host animal""s cells, sporozoites develop into multinucleate meronts, also called schizonts. Each nucleus of the meront develops into an infective body called a merozoite which enters new cells and repeats the process. After a variable number of asexual generations, merozoites develop into either microgametocytes or macrogametes. Microgametocytes develop into many microgametes which, in turn, fertilize the macrogametes. A resistant coat then forms around the resulting zygotes. The encysted zygotes are called oocysts and are shed unsporulated in the feces. Infected birds may shed oocysts in the feces for days or weeks. Under proper conditions of temperature and moisture, the oocysts become infective through the process of sporulation. Susceptible birds then ingest the sporulated oocysts through normal pecking activities and the cycle repeats itself. Ingestion of viable, sporulated oocysts is the only natural means of infection.
Infection with Coccidia results in immunity so that the incidence of the disease decreases over time as members of the flock become immune. This self-limiting nature of coccidial infections is widely known in chickens and other poultry. The immunity conferred, however, is species specific such that introduction of another species of Coccidia will result in a new disease outbreak. In addition, infected birds shed considerable numbers of oocysts into the environment so that the introduction of new, previously uninfected birds may result in an outbreak of the disease.
The oocyst wall of Coccidia provides a highly effective barrier for oocyst survival. Oocysts may survive for many weeks outside the host. In the laboratory, intact oocysts are resistant to extremes in pH, detergents, proteolytic, glycolytic, and lipolytic enzymes, mechanical disruption, and chemicals such as sodium hypochlorite and dichromate.
Two methods are currently used to control coccidiosis in poultry. The first involves control by chemotherapy. Numerous drugs are available for the control of coccidiosis in poultry. Because of the number of species which cause the disease, very few drugs are efficacious against all species, although a single drug may be efficacious against several species. In modern broiler chicken production, for example, administration of drugs to control coccidiosis is routine. The expense for preventative medication against coccidiosis in chickens alone has been estimated to exceed $90 million dollars in the United States and $300 million worldwide.
Three programs of drug administration are commonly used in the domestic poultry industry. The simplest is the continuous use of a single drug from day one until slaughter. The shuttle or dual drug program involves the use of two different drugs, one administered in the xe2x80x9cstarterxe2x80x9d ration and a second drug administered in the xe2x80x9cgrowerxe2x80x9d ration. In the third method, drugs are rotated in hopes of preventing the development of drug resistant strains.
The development of drug resistance by Coccidia is a serious limitation on the effectiveness of chemotherapy to control the disease. Surveys in the United States, South America and Europe have revealed widespread drug resistance in Coccidia. Since drug resistance is a genetic phenomenon, once established, drug resistance can remain in the population for many years until reduced by natural selection pressure and genetic drift.
The use of drugs in animals used for food production is also coming under increasing scrutiny by the public. Consumers are increasingly concerned with the possibility of drug residues in food. This creates pressure in the poultry industry to reduce the use of drugs to control coccidiosis.
Vaccination of birds against Coccidia is an alternative to chemotherapy. An advantage of vaccination is that it can greatly reduce or eliminate the need to administer anti-coccidial drugs, thus reducing costs to poultry producers, preventing the development of drug-resistant strains, and lessening consumer concerns about drug residues.
Numerous methods have been developed to immunize poultry against Coccidia. The successful methods have all been based on the administration of live protozoa, often of an attenuated strain. The most common route of inoculation is oral, although other routes have been used. Edgar, U.S. Pat. No. 3,147,186, teaches vaccination of chickens by oral administration either directly into the mouth or via the feed or water of viable E. tenella sporulated oocysts. Davis et al., U.S. Pat. No. 4,544,548, teaches a method of vaccination by continuous administration of low numbers of sporulated oocysts, with or without simultaneous administration of anti-coccidial drugs.
Oral administration of attenuated strains of sporocysts has also been utilized to confer immunity against coccidiosis. Shirley, U.S. Pat. No. 4,438,097; McDonald, U.S. Pat. No. 5,055,292; and Schmatz et al., PCT publication No. WO 94/16725. An alternative to attenuation is disclosed in Jenkins et al., Avian Dis., 37(1):74-82 (1993), which teaches the oral administration of sporozoites that have been treated with gamma radiation to prevent merogonic development.
Parenteral routes of vaccination have included subcutaneous or intraperitoneal injection of excysted sporozoites, Bhogal, U.S. Pat. No. 4,808,404; Bhogal et al., U.S. Pat. No. 5,068,104, and intra ovo injection of either oocysts or sporocysts, Evans et al., PCT publication No. WO 96/40233; Watkins et al., Poul. Sci., 4(10) :1597-602 (1995). Thaxton, U.S. Pat. No. 5,311,841, teaches a method of vaccination against Coccidia by administration of oocysts or sporozoites to newly hatched chicks by yolk sac injection.
One common factor for all methods of vaccinating poultry against coccidiosis, has been the requirement of viable protozoa in order to confer immunity. Use of non-viable protozoa or antigens from protozoa have routinely been unsuccessful in conferring a high level of immunity. Rose and Long, 18th Symp. Br. Soc. Parasitol., pp. 57-74, 1980. Thus, in any vaccination program for coccidiosis, it is critical that viable protozoa be used.
The requirement for living protozoa to confer immunity necessitates that any preparation of protozoa to be used in anti-coccidial vaccines be tested for viability prior to use. In addition, the availability of a rapid, accurate viability test would be useful in screening potential anti-coccidial drugs.
The traditional method of determining the viability of coccidial protozoa has been to administer the organisms to susceptible hosts and wait to see if clinical symptoms develop. Although gross pathology provides a means to evaluate infection, this method is costly, both in terms of animals used and the time to complete the assay. For example, in chickens, it takes 4-7 days after infection for clinical signs of the disease to manifest themselves.
An alternative method to assess viability is the in vitro infection of cells. For example, Raether et al., Parasitol. Res. 77(5):386-94 (1991), used the ability of E. tenella sporozoites to invade primary chick-kidney cells to assess the effectiveness of salinomycin sodium. Although more economical in terms of animal use, viability assays based on invasion of in vitro cell cultures require days before results can be obtained. In addition, such assays are more qualitative rather than quantitative, since they cannot assay the viability of individual protozoa. What is needed, therefore, is an assay that can rapidly and accurately assess the viability of individual protozoa within a sample.
One method which has been used to assess cell viability in general is the use of vital dyes. Vital dyes are dyes which are able to differentiate between living (vital) and dead (non-vital) cells on the basis of differential staining. One common class of vital dyes relies on membrane integrity to assess viability. Typically with these dyes, viable cells with intact membranes exclude the dye, while cells whose membrane integrity has broken down and so are dead or dying are stained. While there is not an exact equivalence between the presence of an intact cell membrane and the ability of the cell to maintain its existence, it is common in the art to consider cells whose membrane has become irreparably disrupted as dead.
Originally, non-flourescent dyes such a Trypan Blue were used to assess viability. However, due to their greater sensitivity and compatibility with flow cytometers, fluorescent dyes are now commonly used to assess viability based on membrane integrity. Commonly used fluorescent dyes which are excluded from intact cells are polar heterocyclic compounds such as propidium iodide, ethidium bromide and ethidium homodimer.
Viability tests based on differential staining have been applied to a number of cell types and organisms. The use of vital stains and flow cytometry to assess viability of bacteria has been reviewed by Davey and Kell, Microbiol. Rev., 60(4):641 (1996). Differential staining has also been used to assess the viability of a number of cell types including thymocytes (Aeschbacher et al., Cell Biol. Toxicol. 2(2):247-55 (1986)), cartilage and corneas (Yang et al., Cell Transplant. 7(5):443-51 (1998)), pancreatic islet cells (Corominola et al., Cryobiology 37(2):110-18 (1998)), and lymphocytes (Liegler et al., Clin. Diagn. Lab. Immunol. 2(3):369-76 (1995)).
The use of vital dyes to assess the viability of Eimeriina protozoa has been more limited. Vital dyes have been used to assess the viability of excysted stages of Eimeria. Raether et al., Parasitol. Res. 77:386-94 (1991), used a combination of propidium iodide and fluorescein diacetate to determine the viability of E. tenella sporozoites after exposure to salinomycin sodium, an anti-coccidial drug. Brown et al., FEMS Microbiol. Lett., 142(2-3)203-08 (1996) describes a method for assessing the viability of E. tenella sporozoites using acridine orange and bis-benzimide. Geysen et al., J. Parasitol., 77(6):989-993 (1991), used a combination of ethidium bromide and acridine orange to assess the viability of E. tenella merozoites and schizonts isolated from the intestinal mucosa of infected chickens. Davis et al., Mem. Inst. Oswaldo Cruz, 87(Suppl. III):235-239(1992) used the vital dye hydroethidine to measure the viability of the intraerythrocytic form of another member of the class Sporozoea, Babesia bovis. The use of vital dyes has apparently found only limited use in determining the viability of encysted protozoa of the Sporozoea class, no doubt due to resistence of the cyst or outer coating to dye penetration.
A notable exception to this is members of the genus Cryptosporidia. A combination of DAPI (4xe2x80x2-6-diamindino-2-phenylindole) and PI (propidium iodide) has been used by several authors as a means to determine the viability of Cryptosporidia oocysts with varying success. Jenkins et al., Appl. Environ. Microbiol., 63(10):3844-50 (1997) and Campbell et al., Appl. Environ. Microbiol., 58(11):3488-93 (1992) reported a strong correlation between viability estimates based on vital staining and in vitro excystation. Jenkins et al., also reported that Cryptosporidia oocysts classified as viable were able to cause infection, but no direct correlation between infectivity and viability as determined by vital staining was made. The accuracy of viability tests in Cryptosporidia based on differential staining has been questioned. Black et al., FEMS Microbiol. Lett., 135(2-3): 187-89 (1996) reported that both DAPI/PI and in vitro excystation assays overestimate infectivity. The success of vital staining of Cryptosporidia species as opposed to other Eimeriina may be due to difference in the life cycle. Unlike other Eimeriina, Cryptosporidia do not form sporocysts. Instead, the sporozoites form in the oocysts. Regardless of the reason why, there have been no reports of successful application of vital dyes to encysted stages of the genus Eimeria.
The present invention provides a rapid, reliable means to assess viability of protozoa in vaccine preparations prior to either distribution or administration. In addition, the present invention may be used in assessing the sensitivity of protozoa to anti-coccidial drugs, both in terms of new drug development and in terms of choosing appropriate anti-coccidial drugs for administration to animals with clinical coccidial infections. Further, the invention may be used to evaluate the effectiveness of agents for use in disinfecting equipment and environments contaminated by coccidial protozoa.
Among the aspects of the invention, therefore, is a process for determining the viability of sporocyst-forming protozoa. In this process, a sample of sporocysts is obtained from a starting population of protozoa. The starting population can be either a naturally occurring population or one maintained in the laboratory. If the population contains primarily oocysts or sporulated oocysts, the formation of sporocysts can be induced by well established methods. The sporocysts in the sample are treated with at least one vital dye to yield a detectable signal. Staining is then determined using any means suitable for detecting the stained protozoa. By correlation, the viability of the protozoa in the original population can be estimated based on the extent of staining of the protozoa in the sample.
Another aspect of the invention is a method for evaluating the effectiveness of a composition against a sporocyst-forming protozoa. In this method, a first sample containing sporocysts is prepared from a population of sporocyst-forming protozoa and treated with at least one vital dye. The treated sample is then examined for staining and the extent of staining correlated with the viability of the protozoa in the population. The population is then exposed to the composition to be tested, after which a second sample containing sporocysts is obtained. The second sample is also stained with at least one vital dye and the extent of staining examined. The extent of staining in the second sample is correlated with the viability of the protozoa in the population. Thus, by comparing the viability of protozoa in the two samples, the effectiveness of the composition can be determined.
Yet another aspect of the invention is a method for evaluating a means for storing or maintaining sporocyst-forming protozoa. The method comprises, preparing a first sample of sporocysts from a population of sporocyst-forming protozoa and treating the sample with at least one vital dye. The sample is then examined for staining and the extent of staining correlated with the viability of protozoa in the population. The population of protozoa is then stored using the method to be tested and at least on additional sample of sporocysts prepared. The additional sample is treated with at least vital dye and examined for the extent of staining. The extent of staining is then correlated with the viability of the population after storage. The viability of the population before and after storage is then compared to evaluate the storage method.
Still another aspect of the invention is a method for evaluating a management practice for controlling protozoa. The method comprises preparing a first sample of sporocysts obtained from the environment to be managed and treating the sample with at least one vital dye. The extent of staining is then examined and correlated with the viability of protozoa in the environment. The management practice to be evaluated is then implemented. After implementation of the management practice, at least one additional sample of sporocysts from the environment is prepared. The sample is treated with at least one vital dye and examined for the extent of staining. The extent of staining is then correlated with the viability of the protozoa in the environment after implementation of the management practice. By comparing the viability of protozoa in the environment before and after implementation of the management practice, the effectiveness of the practice in controlling protozoa in the environment managed can be assessed.
Yet another aspect of the invention is to assess the viability and hence the infectivity of protozoa found in the environment. The disclosed invention is also useful for assessing the sensitivity of protozoa to anti-protozoal drugs, both in terms of new drug development and in choosing the most effective therapeutic agent for treating animals with protozoal infections. In addition, the invention can be used to evaluate the effectiveness of agents for use in disinfecting equipment and environments contaminated by coccidial protozoa.